Protein Z-dependent protease inhibitor

ABSTRACT

The disclosure describes the purification and isolation of a novel human protein Z-dependent protease inhibitor (ZPI) from plasma characterized as having a molecular weight of about 72 kDa, being a single chain protein with an N-terminal amino acid sequence of LAPSPQSPETPA, and which produces a rapid inhibition of factor Xa in the presence of human protein Z (PZ), calcium ions and cephalin. The disclosure further describes the isolation and cloning of the ZPI cDNA from a human cDNA library. The ZPI cDNA is 2.44 kb in length and has an open reading frame that encodes the 423 residue mature ZPI protein and a 21 residue signal peptide. PZ, ZPI and the combination of PZ and ZPI are used to inhibit blood coagulation.

This is a division of application Ser. No. 09/271,608, filed Mar. 17,1999, now U.S. Pat. No. 6,245,741 issued Jun. 12, 2001 which claimspriority is application Ser. No. 60/086,571, filed May 19, 1998.

This invention was made in part with government support under grantnumbers HL 34462 and HL 60782 awarded by the National Institutes ofHealth. The government has certain rights to the invention.

FIELD OF THE INVENTION

The present invention relates to the field of vitamin K-dependent plasmaproteins such as the four classical clotting factors (Factors II, VII,IX and X), protein C, protein S and, more particularly, to human proteinZ (PZ).

BACKGROUND OF THE INVENTION

[Note: Literature references on the following background information andon conventional test methods and laboratory procedures well-known to theordinary person skilled in the art, and other such state-of-the-arttechniques as used herein, are indicated by numbers in parentheses andappended at the end of the specification.]

Vitamin K is required for the post-translational formation of gammacarboxyglutamic acid (Gla), which is present in a number of plasmaproteins that are involved in coagulation: Prothrombin, factors VII, IX,X, protein C and protein S (1,2). Gla-mediated calcium ion binding inthese proteins is necessary for their association with phospholipidsurfaces and is critical for their hemostatic function (3). In 1977,Prowse and Esnouf identified an additional vitamin K-dependent proteincirculating in bovine plasma and named it protein Z (PZ) (4). Initiallythought to represent a single chain form of bovine factor X, bovine PZwas later identified as a discrete Gla-containing protein (5,6). Thehuman counterpart of bovine PZ was isolated in 1984 (7).

Human PZ is a 62,000 molecular weight glycoprotein that has a plasmahalf-life of ˜2.5 days (8). Plasma PZ levels in blood donors span abroad range with a mean concentration of 2.9±1.0 μg/mL in EDTAanticoagulated samples (corresponding to ˜2.6 μg/mL in citrate plasma)(8). The amino-terminal half of PZ is very homologous (40-50%) to thoseof factors VII, IX and X, and contains a Gla-domain, two EGF-likedomains, and a region which connects to a homologue of the catalyticdomains present in the serine protease zymogens. In the carboxy-terminaldomain of PZ, however, the region around the typical “activation” siteis absent and the His and Ser residues of the catalytic triad arelacking (the Asp residue is conserved) (9,10).

McDonald et al (11) have recently reported that the kinetics of thebinding of human and bovine PZ to phosphatidylcholine/phosphatidylserine(PC/PS=75%/25%) vesicles is different from that of the other vitaminK-dependent coagulation factors. The k_(assn)(10⁻⁵s⁻¹M⁻¹) andk_(dssn)(s⁻¹) rate constants are 1.95 and 0.0063 for bovine PZ and 3.36and 0.057 for human PZ. In comparison the values of these constants forbovine prothrombin are 176.0 and 1.9, respectively. Thus, theassociation and dissociation rate constants for bovine and human PZ aredramatically slower than those of prothrombin and the dissociation ofbovine PZ from phospholipids is significantly slower than that of humanPZ.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to human protein Z (PZ) and a novel human proteinZ-dependent inhibitor (ZPI).

In accordance with one embodiment of the invention, a novel humanprotein Z-dependent protease inhibitor (ZPI) has been purified andisolated from plasma and characterized structurally and biologically.ZPI is a 72,000 molecular weight, single chain protein with an initiallydetermined N-terminal amino acid sequence of LAPSPQSPEXXA(X=indeterminant). Using the conventional three-letter amino acidsymbols required by 37 CFR §1.821-1.825, the N-terminal sequence is asfollows:

[SEQ ID NO:1] Leu Ala Pro Ser Pro Gln Ser Pro Glu Xaa Xaa Ala1               5                   10

This sequence does not match or show significant homology with thesequences accessible in publicly available protein or DNA data bases.Thus, it believed that ZPI is a novel protein.

ZPI has an estimated concentration in citrate plasma of about 1.0 to 1.6μg/mL. In systems using purified components, the factor Xa inhibitionproduced by ZPI is rapid (>95% within one minute by bioassay) andrequired the presence of human protein Z, calcium ions and cephalin. Theinhibitory process appears to involve the formation of a factorXa-PZ-ZPI complex at the phospholipid surface.

To further characterize ZPI in another embodiment of the invention, itscDNA was isolated and cloned from a human liver cDNA library. The ZPIcDNA is 2.44 kb in length and has a relatively long 5′ region (466 nt)that contains six potential ATG translation start codons. ATG's 1 to 4are followed by short open reading frames, whereas ATG₅ and ATG₆ are inan uninterrupted 1335 bp open reading frame that includes the encodedZPI protein. The deduced ZPI protein of 444 amino acids has a typical 21residue signal peptide that is followed by the N-terminal sequence ofthe purified protein in which the initially indeterminate residues 10and 11 in SEQ ID NO:1 are, respectively, threonine and proline, as inSEQ ID NO: 8.

In vitro experiments show that ATG₆ is sufficient for the expression ofrZPI in cultured Chinese hamster ovary (CHO) cells. Northern analysissuggests that the liver is a major site of ZPI synthesis.

The predicted 423 residue amino acid sequence of the mature ZPI proteinis 25-35% homologous with members of the serpin superfamily of proteaseinhibitors and is 78% identical to the amino acid sequence predicted bya previously described cDNA isolated from rat liver,regeneration-associated serpin protein-1 (rasp-1).

Alignment of the amino acid sequence of ZPI with those of other serpinspredicts that Tyr387 (Y387) is the P₁ residue at the reactive center ofthe ZPI molecule. Consistent with this notion, rZPI (Y387A), an alteredform of ZPI in which tyrosine 387 has been changed to alanine, lacksPZ-dependent factor Xa inhibitory activity.

In still other embodiments of the invention, PZ, ZPI and the combinationof PZ and ZPI are used as inhibitors of blood coagulation. Asillustrated below, this is the first work showing that PZ and ZPIproduce inhibition of coagulation. This work also shows that PZ caninhibit coagulation in the absence of ZPI (FIG. 5).

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming theinvention, it is believed that the invention will be better understoodfrom the following preferred embodiments of the invention taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation which shows the effect of PZ on theinhibition of factor Xa by antithrombin III. Reactions containing factorXa (5 nM), CaCl₂ (4 mM), with or without PZ (40 nM), and with or withoutcephalin (15 μM) were incubated 5 min. at 22° C. before the addition ofantithrombin III (3.4 μM). At the indicated times thereafter, sampleswere removed, diluted in HSA with 1 mM EDTA, and assayed for factor Xaactivity by bioassay.

(▾), without cephalin, without PZ;

(∘), without cephalin, with PZ;

(), with cephalin, without PZ;

(), with cephalin, with PZ.

FIG. 2 is a graphical representation which shows the factor Xainhibition by serum. Factor Xa (5 nM), CaCl₂(4 mM), cephalin (15 μM)with or without PZ (40 nM) were incubated 5 min at 22° C. before theaddition of barium absorbed serum (25% v/v) which had been previouslytreated for 30 min. with rabbit preimmune or immune anti-ZPI IgG (300μg/mL). At the specified times thereafter, samples of the reactions werediluted in HSA with 1 mM EDTA and assayed for factor Xa activity bybioassay.

(), with PZ and preimmune IgG;

(▴), with PZ and immune IgG;

(∘), without PZ and with preimmune IgG;

(▾), without PZ and with immune IgG.

FIG. 3 shows the SDS-Page of purified ZPI. ZPI (5 μg) was analyzed with(lane 2, right) or without (lane 1, left) reduction with 5%2-mercaptoethanol. Protein was stained with Coumassie Brilliant Blue.The position of molecular weight standards in kDa is shown on the left.

FIG. 4, in four parts, FIGS. 4A, 4B, 4C and 4D, is a graphicalrepresentation which shows the PZ-dependent inhibition of factor Xa byZPI.

FIG. 4A shows the ZPI dose/response. Reactions containing factor Xa (2.5or 5.0 nM), CaCl₂ (4 mM), cephalin (15 μM) and PZ,(40 nM) were incubatedwith increasing concentrations of ZPI for 15 min. at 22° C. beforeremaining factor Xa activity was determined by amidolytic assay. Themolar concentration of ZPI was estimated assuming 1.0 mg/mL ZPI producesan A₂₈₀ of 1.0. (□), factor Xa (2.5 nM; (∘), factor Xa 5.0 nM.

FIG. 4B shows the PZ dose/response. Reactions containing factor Xa (2.5or 5.0 nM), CaCl₂ (4 mM), cephalin (15 μM) and ZPI (10 nM) wereincubated with increasing concentrations of PZ for 15 min. at 22° C.before remaining factor Xa activity was determined by amidolytic assay.(□), factor Xa 2.5 nM; (∘), factor Xa 5.0 nM.

FIG. 4C shows the cephalin dose/response. Reactions containing factor Xa(2.5 nM), CaCl₂ (4 mM), PZ 40 nM), and ZPI 10 nM) were incubated withincreasing concentrations of cephalin for 15 min. at 22° C. beforeremaining factor Xa activity was determined by amidolytic assay.

FIG. 4D shows time course of factor Xa inhibition by PZ/ZPI. Reactionscontaining factor Xa (5.0 nM), with or without CaCl₂ (4 mM), with orwithout cephalin (15 μM), and with or without PZ (40 nM) were incubated5 min. at 22° C. before the addition of ZPI (10 nM). At the specifiedtimes thereafter remaining factor Xa activity was determined by bioassayor amidolytic assay. Bioassay: (), with all reactants. Amidolyticassay: (), with all reactants; (▴), without CaCl₂; (▾), withoutcephalin; (♦), without PZ.

FIG. 5 is a graphical representation which shows the effect of anti-ZPIantibodies on factor Xa-induced coagulation of plasma. Reactions (200μL) containing factor Xa (0.125 nM), CaCl₂ (5 mM), cephalin (18.75 μM),with or without PZ (50 nM) were incubated in the sample cup of afibrometer. After 2 min. at 37° C., 50 μL of factor X deficient plasmawhich had been treated with rabbit preimmune IgG (600 μg/mL) or immuneanti-ZPI IgG at increasing concentrations for 30 min. was added and theclotting time measured. Apparent factor Xa inhibition (76%) produced bythe inclusion of PZ during the preincubation period and using plasmatreated with preimmune IgG is listed as maximal PZ-dependent inhibition(100% on the ordinate). The concentration of Anti-ZPI IgG used to treatthe plasma is listed on the abscissa.

FIG. 6 shows the nucleotide sequence (SEQ ID NO: 7) and deduced aminoacid sequence (SEQ ID NO: 8) of human ZPI CDNA. The amino acid sequenceis shown in single letter code beneath the nucleotide sequence.Nucleotide/amino acid numbers are shown in the column at the left.Translation is depicted as starting at ATG₆(nt 467). An alternativeinitiation codon, ATG₅(nt 347), is underlined in dashes (see ExampleII). Amino acid sequences derived from purified plasma ZPI areunderlined. N* denote potential sites of N-linked glycosylation and thetyrosine residue at the putative P₁ site at the reactive center of ZPIis shown in bold print. The amino acid sequence is shown in three lettercode in the attached Sequence Listing.

FIG. 7 shows the Northern analysis of multiple tissues for ZPI mRNA. ANorthern blot nitrocellulose membrane containing 2 μg poly A⁺mRNA fromvarious human tissues in each lane was hybridized with a ³²P-labeledfull length ZPI CDNA probe (above) or a ³²P-labeled β-actin cDNA probe(below).

FIG. 8 shows the alignment of the C-terminal amino acid sequences of ZPI[SEQ ID No: 9] and other serpins. Amino acid sequences of rat rasp-1(RASP-1) [SEQ ID No: 10] and human α₁-antitrypsin (A1AT) [SEQ ID No:11], antitrypsin related sequence (A1AU) [SEQ ID No: 12], antithrombin(AT-III) [SEQ ID No: 13], heparin cofactor II (HC-II) [SEQ ID No: 14],and protease nexin 1 (PN-1) [SEQ ID No: 15] were extracted from theGenBank data base (accession numbers 2143953, 1703025, 112891, 113936,123055, and 121110, respectively). Identical amino acids are darklyshaded. The arrowhead indicates the column containing the P₁, residue atthe reactive center of each serpin.

FIG. 9 shows the Western blot analysis of wild-type and altered forms ofrecombinant ZPI. Serum-free conditioned media (10 μL) fromnon-transfected CHO cells (CHO) and CHO cells expressing rZPI(WT) (Tyr),rZPI(Y387A)(Ala) and rZPI(Y387R) (Arg) were analyzed by 12% SDS-PAGE andWestern blotting using a mouse monoclonal anti-ZPI antibody. Themigration of pre-stained molecular weight standards listed in kDa isdepicted on the left. Below the blot is listed the ZPI activity of eachconditioned media (average of duplicate measurements). The protein bandidentified at ˜54,000 is detected in the conditioned media ofnon-transfected CHO cells and appears to be unrelated to rZPI.

In order to illustrate the invention in greater detail, the followingspecific laboratory examples were carried out. Although specificexamples are thus illustrated herein, it will be appreciated that theinvention is not limited to these specific, illustrative examples or thedetails therein.

EXAMPLE I Purification and Isolation of ZPI from Human Plasma

MATERIALS and METHODS

Materials. Sodium dodecyl sulfate (SDS), HEPES, MES, Trizma Base,Diisopropylfluorophosphate (DFP), Triton X-100, Tween 20,ethylenediaminetetraacetic acid (EDTA), polyethylene glycol (8,000 MW) SFast Flow, bovine serum albumin, and rabbit brain cephalin were fromSigma Chemical Co. (St. Louis, Mo.). Mono-Q, Monol-S andheparin-Sepharose were purchased from Pharmacia Biotech (Piscataway,N.J.). Low molecular weight standards for polyacrylamide gelelectrophoresis were from Bio-Rad Laboratories (Richmond, Calif.) andprotein A-agarose from Repligen (Cambridge, Mass.). Spectrazyme Xa(MeO-CO-D-CHG-Gly-Arg-pNA.AcOH) was from American Diagnostica, Inc.(Greenwich, Conn.). Plasma/serum. Factor X deficient plasma waspurchased from George King Biomedical (Overland Park, Kans.). To produceserum, fresh blood was allowed to clot for one hour at 37° C., the clotrimmed, and serum collected following centrifugation (10,000 g, 20 min).Barium-absorbed serum was produced by adding sodium oxalate (10 mMfinal) and two subsequent absorptions with barium sulfate (100 mg/mL, 4°C., 30 min). By monoclonal antibody sandwich immunoassay (8), the bariumabsorbed serum contained 0.10 μg/mL PZ.

Proteins. Alpha-1-antitrypsin was purchased from Sigma,alpha-2-antiplasmin and thrombin from American Diagnostica, Inc.,protein C inhibitor (PCI) from Enzyme Research Laboratories (South Bend,Ind.), and antithrombin III from Chromogenix AB (Molndal, Sweden).Alpha-2-macroglobulin was a gift from A. Schwartz (WashingtonUniversity, St. Louis) and heparin cofactor II a gift from D. Tollefsen(Washington University). Inter-alpha-trypsin inhibitor was purified aspreviously described (12). Prothrombin and factor X were purified andfactor Xa was produced from purified factor X using insolubilizedX-coagulant protein from Russells viper venom as previously described(13). Additional factor Xa was purchased from Enzyme ResearchLaboratories.

PZ was isolated from citrated fresh frozen plasma (Missouri-IllinoisRegional Red Cross) using a four step purification procedure thatincluded barium citrate absorption-elution, ammonium sulfatefractionation, monoclonal antibody anti-PZ immunoaffinity chromatographyand Mono-Q anion exchange chromatography. Thrombin-cleaved PZ (PZ_(T))was produced by incubating 1 mg/mL PZ with 300 U/mL thrombin in 0.1 MNaCl, 0.05 M Tris-HCl, pH 8.0 for 3 hours at 37° C. The solution wastreated with DFP (5 mM final) before removing the thrombin by passagethrough a small column of CG-50 in the same buffer (7). The N-terminalamino acid sequence of PZ_(T) (˜56,000 MW) (7) after SDS-PAGE, transferto a PVDF-Plus membrane (Micron Separations, Inc., Westborough, Mass.)and gas-phase sequencing (Applied Biosystems, Foster City, Calif.) bythe Protein Chemistry Laboratory (Washington University) isRYKGGSPXISQPXL (X=indeterminant). Using the conventional three-letteramino acid symbols required by 37 CFR §1.821-1.825, this sequence is asfollows:

Arg Tyr Lys Gly Gly Ser Pro Xaa Ile Ser Gln Pro Xaa Leu 1 5 10

This amino acid sequence is identical to the sequence of PZ beginning atresidue 44 of the mature protein. Thus, thrombin appears to cleave PZfollowing Arg43 thereby separating the Gla-domain from the remainder ofthe molecule. One-stage, factor Xa-induced coagulation assay. Cephalin(75 μM) 50 μL, 50 μL CaCl₂ (25 mM), 50 μL PZ or PZ_(T) (160 nM) and 50μL factor Xa (0.5 nM) are incubated at 37° C. in the sample cup of afibrometer (BBL, Cockeysville, Md.). After 2 minutes, 50 μL factor Xdeficient plasma is added and the clotting time measured. In certaintests the PZ or factor Xa were added at varying times during thepreincubation period and the cephalin or PZ reagents were added to thereaction with the factor X deficient plasma (100 μL of 1:1 mixtures).Apparent factor Xa activity is determined by comparing the clotting timewith a standard curve constructed using various concentrations of factorXa in the absence of PZ.

Factor Xa bioassay. Cephalin (75 μM) 50 μL, 50 μL CaCl₂ (25 mM), and 50μL buffer containing 0.1 M NaCl, 0.05 M HEPES, pH 7.4, and bovine serumalbumin (1 mg/mL) (HSA) are incubated at 37° C. After 30 seconds, 50 μLof the sample diluted in HSA with 1 mM EDTA is added followedimmediately by 50 μL of factor X deficient plasma. Apparent factor Xaactivity is determined by comparing the clotting time with a factor Xastandard curve.

Factor Xa amidolytic assay. Mixtures (100 μL) containing variousconcentrations of cephalin, PZ, ZPI, factor Xa and Ca++ ions in HSAbuffer are incubated at 22° C. in the wells of a microtiter plate. Afterthe specified period of time, 50 μL of Spectrazyme Xa (0.5 mM) is addedand the initial rate of substrate cleavage (A₄₀₅/min.) determined in aVmax microtiter plate reader (Molecular Devices, Menlo Park, Calif.). Intests studying the time course of factor Xa inhibition by PZ/ZPI, thesolution containing the Spectrazyme Xa (0.5 mM) also contained 15 mMEDTA and 0.3 M Tris-HCl, pH 8.3. Factor Xa activity is determined bycomparing the initial rate of substrate cleavage with a standard curveproduced with various concentrations of factor Xa in the same bufferconditions.

Two-stage factor Xa inhibition assay. To measure ZPI functional activity10 μL cephalin (75 μM), 10 μL CaCl₂ (25 mM), 10 μL PZ (200 nM), 10 μL ofthe sample to be tested diluted in HSA, and 10 μL factor Xa (2.5 nM) areincubated in the sample cup of a fibrometer at 37° C. After 60 seconds,50 μL HSA, 50 μL cephalin (75 μM), 50 μL CaCl₂ (25 mM) and 50 μL factorX deficient plasma are added in succession and the clotting timemeasured. ZPI activity is determined by comparing the clotting time witha standard curve produced using various concentrations of purified ZPI.The activity of 1 μg purified ZPI was arbitrarily assigned a value of 1unit.

Purification of ZPI. Human citrate fresh frozen plasm (2.3 liters) fromthe Missouri-Illinois Regional Red Cross was thawed at 37° C. andtransferred to the cold room. A six-step purification was carried out asfollows:

1. Barium citrate adsorption and ammonium sulfate fractionation (4° C.).230 mL of 1.0 M BaCl₂ was added dropwise over 45 minutes and the mixturewas stirred an additional 15 min. The barium citrate precipitate wasremoved by centrifugation at 3,000 g for 20 min. and supernatant plasmacollected. Ammonium sulfate was added to 45% saturation and the mixturestirred for 30 min. before the precipitate was removed by centrifugationat 10,000 g for 20 min. Sufficient ammonium sulfate was added to bringthe supernatant to 75% saturation and the mixture stirred for 30 min.before centrifugation at 10,000 g for 20 min. The protein precipitatewas dissolved in 0.1 M NaCl, 0.05 M Tris-HCl, pH 7.5, treated with DFP(1 mM) and dialyzed overnight against the same buffer.

2. Polvethylene glycol (PEG) fractionation (22° C.). Sufficient 50% w/vPEG (8,000 MW) was added dropwise to the sample to produce a PEGconcentration of 7.5% and the mixture stirred for 30 min. before theprecipitate was removed by centrifugation at 10,000 g for 20 min. PEG(50% w/v) was added dropwise to the supernatant solution to produce aPEG concentration of 18.5% and the mixture stirred for 30 min. beforecentrifugation at 10,000 g for 20 min. The protein precipitate wasdissolved in 0.1 M NaCl, 0.020 M MES, pH 6.15 and treated with DFP (1mM).

3. S Fast Flow cation exchanae chromatography (4° C.). The sample wasapplied at flow rate of 150 mL/hr to a 5×47 cm. column of S Fast Flowequilibrated in 0.1 M NaCl, 0.02 M MES, pH 6.15. The column was washedwith 1.5 L of the same buffer and then eluted with a linear gradient to0.5 M NaCl, 0.02 M MES, pH 6.15 over 8 L. Fractions containing ZPIactivity, which eluted at ˜0.25 M NaCl, were combined and the poolconcentrated to 25 mL (YM 10, Amicon, Danvers, Mass.) and treated withDFP (5 mM).

4. Mono-O anion exchange chromatography (22° C.). The concentratedsample was diluted 2.5-fold with 0.02 M MES, pH 6.15 and applied at aflow rate of 1.5 mL/min. to a 10 mL Mono-Q column equilibrated in 0.1 MNaCl, 0.02 M MES, pH 6.15 containing 0.1% (v/v) Tween-20. The column waswashed with 15 mL of the same buffer and then eluted with a lineargradient to 0.5 M NaCl in the same buffer over 100 mL. Fractionscontaining ZPI activity, which eluted at ˜0.18 M NaCl were combined andtreated with DFP (5 mM).

5. Heparin-Sepharose affinity chromatography (22° C.). The sample wasdiluted 2-fold with 0.02 M MES, pH 6.15 and applied at a flow rate of 1mL/min. to a 5 mL heparin-Sepharose column equilibrated in 0.1 M NaCl,0.02 M MES, pH 6.15 containing 0.1% (v/v) Tween-20. The column waswashed with 10 mL of the same buffer and then eluted with a lineargradient to 0.6 M NaCl in the same buffer over 50 mL. Fractionscontaining ZPI activity, which eluted at ˜0.25 M NaCl, were pooled andtreated with DFP (5 mM).

6. Mono-S cation exchange chromatography (22° C.). The sample wasdiluted 3-fold with 0.02 M MES, pH 6.15 and applied at a flow rate of0.5 mL/min. to a 1 mL Mono-S column equilibrated in 0.1 M NaCl, 0.02 MMES, pH 6.15 containing 0.01% (v/v) Tween-20. The column was washed with2 mL of the same buffer and then eluted with a linear gradient to 0.5 MNaCl in the same buffer over 20 mL. Fractions containing ZPI activity,which eluted at ˜0.25 M NaCl, were pooled and the purified ZPI stored at−70° C. in small aliquots. The molar concentration of ZPI was estimatedassuming a ZPI concentration of 1.0 mg/mL produces an absorbance of 280nm (A₂₈₀) of 1.0 and a molecular weight of 72,000.

The foregoing six-step purification of ZPI is summarized in Table II,below.

Other Methods. SDS polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed using the method of Laemmli (14). Rabbit polyclonal anti-ZPIantibodies were developed as previously described using purified ZPI asimmunogen (15). Pre-immune and immune IgG were isolated using proteinA-agarose. N-terminal amino acid sequence analysis of purified ZPI wasperformed by the Protein Chemistry Laboratory (Washington University)using a gas-phase sequenator (Applied Biosystems). Two separate analyseswere performed with 0.3 nmol of ZPI and gave identical results. Thephospholipid content of the rabbit brain cephalin was determined asinorganic phosphate (16).

RESULTS

Reduction in Factor Xa Procoagulant Activity in the Presence of PZ, Ca++Ions and Phosphopids.

Initial studies investigating the potential function of human PZ showedthat the apparent procoagulant activity of factor Xa in a one-stageplasma coagulation assay was reduced if the factor Xa was firstincubated with PZ (Table I). The inhibitory effect was time dependent,required the presence of calcium ions and procoagulant phospholipids(rabbit brain cephalin), and appeared predominantly related to theperiod of preincubation of PZ with phospholipids (Table I). Thrombintreatment of PZ, which cleaves PZ at Arg43 and separates the Gla domainfrom the remainder of the molecule (see Methods), abolished theinhibitory effect. These results suggest that an interaction betweenfactor Xa and PZ may occur at the phospholipid surface. Consistent withthis belief, the rate of inhibition of factor Xa by antithrombin III wasslowed by PZ in the presence bf cephalin and Ca++ ions (t_(½) 35 min.vs. 15 min.) (FIG. 1).

To further evaluate the effect of PZ on factor Xa inactivation, the timecourse of the loss of factor Xa activity in PZ-depletdd serum with andwithout added PZ in the presence of cephalin and CaCl₂ was determined bybioassay (FIG. 2). An additional early loss of factor Xa activity isdemonstrable in the presence of PZ suggesting that serum contains aPZ-dependent factor Xa inhibitor(s). However, the curves describing theloss of factor Xa activity in serum in the presence and absence of PZintersect so that ultimately the factor Xa activity remaining in serumwith PZ is greater than that in serum without PZ. In systems containingpurified proteins, PZ does not enhance the inhibition of factor Xa byalpha-1-antitrypsin, protein C inhibitor, alpha-2-antiplasmin, heparincofactor II, inter-alpha-trypsin inhibitor or alpha-2-macroglobulin.

Isolation of ZPI

A two-stage bioassay designed to measure PZ-dependent factor Xainhibition was used to isolate a PZ-dependent protease inhibitor (ZPI)from plasma (Methods, Table II). The ZPI activity of the starting plasmacould not be measured due to thrombin generation and fibrin formationduring the first stage of the two-stage factor Xa inhibition assay.Nevertheless, assuming a 50%-75% recovery of ZPI following ammoniumsulfate fractionation of plasma (Table II), it is estimated that theplasma concentration of ZPI is 1.0-1.6 μg/mL (14-22 nM).

By SDS-PAGE analysis, ZPI migrates as a single chain protein with anapparent molecular weight of 72 kDa (FIG. 3). Preliminarycharacterization of the purified protein shows that ZPI activity isabolished by treatment with SDS (1%), urea (8 M), and 2-mercaptoethanol(5% v/v), but is stable in Tween-20 (2%) and Triton X-100 (2%). ZPI isalso unaffected by methylamine treatment under conditions thatcompletely inactivate alpha-2-macroglobulin. The N-terminal amino acidsequence of ZPI is LAPSPQSPEXXA (X=indeterminant), SEQ ID NO: 1. Thissequence does not match nor show significant homology with the sequencesaccessible in publicly available protein or DNA data bases. Thus, ZPImay represent a previously unidentified gene-product.

PZ-dependent Inhibition of Factor Xa by ZPI.

To further investigate the factor Xa-ZPI interaction, mixturescontaining factor Xa, CaCl₂, cephalin and PZ were incubated withincreasing concentrations of ZPI for 15 minutes (22° C.) (FIG. 4A). Theremaining factor Xa activity was then determined in an amidolytic assay(Methods) following the addition of Spectrazyme Xa. The results suggesta high affinity interaction between ZPI and factor Xa with astoichiometry of 1.2:1 (ZPI:factor Xa). Even at relatively highconcentrations of ZPI, however, a significant amount (˜20%) of factor Xaamidolytic activity persists.

The PZ-dose/response of ZPI-mediated factor Xa inhibition was evaluatedin similar reactions (FIG. 4B). Again an apparent stoichiometricrelationship between PZ and factor Xa was demonstrated with a molarratio of 1.4:1 (PZ:factor Xa). Optimal PZ-dependent inhibition of factorXa by ZPI occurs at concentration of mixed rabbit brain phospholipids(cephalin) of ≧15 μM (FIG. 4C). The inhibition of factor Xa produced byZPI is rapid following the incubation of factor Xa with PZ, Ca++ ionsand cephalin (FIG. 4D). Maximal factor Xa inhibition as assessed byamidolytic assay (70%) and bioassay (97%) is reached within <1 minute.Using the amidolytic assay (FIG. 3D) or bioassay, no factor Xainhibition occurs if PZ, phospholipids, or Ca++ ions (1 mM EDTA) isomitted from the reactions.

Serum and plasma were treated with rabbit polyclonal anti-ZPI antibodiesto determine the contribution of serum ZPI to the early, enhancedinhibition of factor Xa produced in the presence of PZ and thecontribution of plasma ZPI to the apparent reduction in factor Xaactivity produced by its incubation with PZ, phospholipids and Ca++ ionsprior to the one-stage bioassay. Treatment with anti-ZPI antibodiescompletely abrogated the PZ-dependent factor Xa inhibition in serum(FIG. 2), but reduced the PZ-mediated inhibitory effect in the plasmaone-stage bioassay by only ˜50% (FIG. 5).

Despite its isolation several years ago, the physiologic function of PZheretofore has remained uncertain. The results in Example I show that PZslows the inhibition of factor Xa by antithrombin III in the presence ofphospholipids and Ca++ ions, but also plays an important role in theinhibition of factor Xa by a novel plasma protein that is herein termedprotein Z-dependent protease inhibitor, ZPI. PZ and/or its apparentinteraction with factor Xa, however, may serve other functions. In thisregard, it is important to note that inhibition of ZPI in the substrateplasma of the one-stage coagulation assay reduced the apparentinhibitory effect of the preincubation of PZ with phospholipids, Ca++ions, and factor Xa by only ˜50%.

The remaining PZ-mediated inhibitory effect could be related to itsinterference with the binding of other proteins to phospholipids (e.g.prothrombin), the slow dissociation of factor Xa from a putative factorXa-PZ-phospholipid-Ca++ complex, or the presence of additionalPZ-dependent coagulation inhibitors in plasma. The relatively slowassociation of PZ with phospholipids (11) is consistent with thetime-dependent inhibitory effect of PZ during its incubation withphospholipids and Ca++ ions in the one-stage assay and, moreover,presumably also explains the absence of clotting time prolongation whenPZ is added instead with the substrate plasma to the reaction.

The inhibition of factor Xa by presumed physiologic concentrations ofZPI requires the presence of phospholipids, Ca++ ions and PZ and appearsto involve a stoichiometric complex of factor Xa, PZ and ZPI at thephospholipid surface. The apparent inhibition of factor Xa produced byZPI, however, is considerably less when remaining factor Xa activity ismeasured using a small molecular weight chromogenic substrate(Spectrazyme Xa), than when remaining factor Xa activity is measured bybioassay. The cause of this discrepancy is not clear.

One explanation of the discrepancy could be the presence in the factorXa preparations of degraded forms of factor Xa that retain activityagainst the chromogenic substrate but do not bind phospholipid and thusare not inhibited by ZPI and lack procoagulant activity. The disparitybetween the inhibitory effect measured by amidolytic assay and bioassay,however, was consistently seen with several factor Xa preparations whoseamidolytic activity was bound by barium sulfate (>97%) and which bySDS-PAGE analysis contained a spectrum of ratios of α and β forms offactor Xa (α:β=1:1 to 1:4) and <5% additionally degraded factor Xa.Moreover, the chromogenic activity of the factor Xa preparations wasinhibited >99% by tissue factor pathway inhibitor (TFPI), suggestingthat the residual chromogenic activity remaining following theinteraction of factor Xa with ZPI was not due to a contaminatingprotease.

EXAMPLE II Isolation and Cloning of ZPI cDNA

MATERIALS AND METHODS

Materials: Fresh frozen human plasma was purchased from the Regional RedCross (St. Louis, Mo.). Multiple human tissue RNA blot and human adultlive cDNA library were from Clontech (Palo Alto, Calif.); human fetalliver CDNA library was from Strategene (La Jolla, Calif.);nitrocellulose membrane from Schleicher & Schuell, Inc. (Keene, N.H.);PVDF membrane from Micron Separations, Inc. (Westborough, Mass.); ³²P-αdATP from NEN Life Scientific, Inc. (Boston, Mass.); and dNTPs fromPharmacia Biotech, Inc. (Piscataway, N.J.). Taq DNA polymerase, DMEMculture medium, fetal calf serum and LipofectAMINE were from Gibco BRL,Life Technologies (Gaithersburg, Md.). Antibiotic G418 (Geneticin) waspurchased from Mediatech, Inc. (Herndon, Va.). Chinese hamster ovary(CHO) cells were from the ATCC (Manassas, Va.). ITS+3 media supplement,protease inhibitor cocktail, soybean trypsin inhibitor, aprotinin, andrabbit brain cephalin were from Sigma Chemical (St. Louis, Mo.). FactorX deficient plasma was from George King Biomedical, Inc. (Overland Park,Kans.). Prestained molecular weight standards for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchasedfrom Bio-Rad (Hercules, Calif.). Proteins: PZ and ZPI were purified fromhuman plasma as previously described (17). A mouse monoclonal anti-ZPIantibody (MC4249.2) was produced using established and previouslyreported techniques (18).

N-terminal amino acid sequencing of ZPI and trypsin-treated ZPI: Samplescontaining 20 μg ZPI or 20 μg ZPI that had been digested with trypsin(1:200 w/w) for 30 minutes at 22° C. were reduced with 2-mercaptoethanol(5%) and separated by 12% SDS-PAGE and electro-transferred to a PVDFmembrane. The membrane was stained for 10 minutes with 0.025% CoomassieBrilliant Blue R-250 in 10% methanol/7% acetic acid, washed withdistilled water and allowed to air dry. Discernible protein bands ofapparent molecular weight 72,000 for ZPI and 43,000 and 41,000 fortrypsin-treated ZPI were cut from the membrane and sequenced by theProtein Chemistry Laboratory (Washington University, St. Louis, Mo.).

ZPI CDNA cloning: The N-terminal amino acid sequence of the two trypticpeptides derived from ZPI was highly homologous to the amino acidsequence predicted by the previously reported cDNA for ratregeneration-associated serpin (rasp-1, GeneBank Accession No.2143953(see Results) (19). Nucleotide sequences derived from rasp-1cDNA,

496-518 (ACCCAGGGTA GCTTTGCCTT CAT), SEQ ID NO: 3, and

805-825 (GTACATCATG GGCACCTTAA C), SEQ ID NO: 4,

were used as the basis for 5′- and 3′-primers, respectively, in a PCRreaction to amplify a DNA fragment from a human fetal liver cDNA library(Strategene). The PCR product, ˜330 bp, was cloned into pGEM-T Easy(Promega, Madison, Wis.) and was found to be 80% homologous with rasp-1cDNA by sequence analysis. Following radiolabeling with ³²P-α dATP andrandom priming, the PCR product was used as a probe to screenapproximately 2×10⁶ plaque-forming units from a human liver cDNA library(Clontech). Hybridization was performed at 42° C. in 5×SSPE,5×Denhardt's solution, 1% SDS, 50% formamide, and 100 μg/mL denaturedsalmon sperm. Filters were washed with 1×SSC and 0.1% SDS solution atroom temperature for 15 minutes and then washed three times with thesame solution at 65° C. for 30 minutes. The twenty-one positive clonesthat remained after plaque purification contained cDNAs of fourdifferent lengths. A representative of the longest cDNA was sequenced inits entirety in both directions.

Northern blot analysis: ³²P-labeled full length ZPI cDNA was used as aprobe for analysis of a human multiple tissue Northern blot membranefrom Clontech (Palo Alto, Calif.) containing 2 μg poly A+ RNA persample. Hybridization was performed under the stringent conditionssuggested by the manufacturer; autoradiography was allowed to proceedovernight.

In vitro expression of wild-type and altered forms of recombinant ZPI(rZPI): A 2.2 fragment of the ZPI cDNA was produced by treatment withSac 1 and Hind III and inserted into the multiple cloning site ofpBluescript KS II. This fragment contained part of the 5′ untranslatedregion, the entire open reading frame,.and the 3′ untranslated region ofthe ZPI cDNA. A 2.3 kb fragment of pBluescript-ZPI was released by PvuII treatment and inserted by blunt-end ligation into the EcoR V site ofthe expression vector pCMV (20) producing pCMV-ZPI(WT). This 2.3 kb DNAfragment contained: 1) ZPI cDNA beginning 120 bp upstream of ATG₆ andlacking the remainder of the 5′ untranslated ZPI CDNA includingATG₁-ATG₅; 2) the coding and 3′ untranslated regions of ZPI cDNA; and 3)˜200 bp of pBluescript KS II DNA. In pCMV, expression is driven by thecytomegalovirus early promoter/enhancer.

PCR-based site-directed mutagenesis with pCMV-ZPI(WT) as template wasused to change the codon for Y387 (TAT) in ZPI to that for alanine (GCT)or arginine (CGT). Mutations were confirmed by sequencing the ZPI cDNAbetween Nsi I (nt 1544) and Spe I (nt 1944) restriction sites which areupstream and downstream of the mutation site. These fragments were theninserted in pCMV-ZPI(WT) at Nsi I - Spe I to produce pCMV-ZPI(Y387A) andpCMV-ZPI(Y387R). pCMV-ZPI(WT), pCMV-ZPI(Y387A) and pCMV-ZPI(Y387R) wereco-transfected with pSV2neo into CHO cells using LipofectAMINE (GIBCOBRL) according to the manufacturer's instructions. Cell clones resistantto G418 were picked at three weeks and expanded. Non-transfected CHOcells and stable CHO clones expressing rZPI(WT), rZPI(Y387A), andrZPI(Y387R) were cultured in 5% CO₂ with DMEM and 10% fetal calf serumin six well culture plates (Costar, Corning, Inc., Corning, N.Y.). Afterthe cells reached confluence, the media was removed and the cells werewashed three times with 5 mL of DMEM before 1 ml of serum-free mediaconsisting of DMEM with ITS+3 media supplement (insulin, transferrin,selenium, Sigma) was added to each well. After an additional 48 hrs. ofculture, the conditioned media was collected, centrifuged (14,000×g×30sec.) to remove cell debris, and analyzed by Western blotting and ZPIfunctional assay. In some experiments, aprotinin (1 μg/mL) and soybeantrypsin inhibitor (2.5 μg/mL) were included in the serum-free media anda 1:10 dilution of protease inhibitor cocktail (Sigma) containing4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF)(100 mM), pepstatin A(1.5 mM), trans-expoxysucciniyl-L-leucyl-amido (4-guanidino)butane(E-64)(1.4 mM), bestatin (4 mM), leupeptin (2.2 mM), and aprotinin (80μM) was added to the conditioned media at the time of its collection.

ZPI functional assay: A two-stage factor Xa inhibition assay was used tomeasure ZPI functional activity as previously described (17). Twenty ALrabbit brain cephalin (75 μM), 20 μL CaCl₂ (25 nM), 20 μL PZ (200 nM) or20 μL HSA (0.1 M NaCl, 0.05 M Hepes, pH 7.4, with 1 mg/mL bovine serumalbumin), 20 μL of the sample to be tested, and 20 μL factor Xa (1 nM)were incubated in the sample cup of a fibrometer at 37° C. After 60sec., 50 μL cephalin (75 μM), 50 μL CaCl₂(25 mM), and 50 μL factorX-deficient plasma were added in succession and the clotting timemeasured. ZPI activity was determined by comparing the clotting timewith a standard curve produced by using various concentrations ofpurified ZPI derived from plasma. One μg of purified plasma ZPI wasdefined to possess 1000 milliunits (mU) of activity.

Western Blotting: SDS-polyacrylamide gel electrophoresis, theelectro-transfer of proteins to nitrocellulose, and incubation of theblot with the monoclonal anti-ZPI antibody (MC4249.2) (10 μg/mL) wereperformed using previously described methods (21). Antibody binding tothe blot was detected using horseradish peroxidase-labeled goatanti-mouse IgG antibodies (Sigma) and enhanced chemoluminescence (ECL)with Super Signal substrate (Pierce, Rockford, Ill.).

RESULTS

Isolation and Secuence of ZPI cDNA

In a search of publicly available protein and DNA databases, theN-terminal acid sequence of ZPI isolated from human plasma, LAPSPQSPEXXA(X=indeterminate), SEQ ID NO: 1, did not show significant sequencehomology with previously reported gene products. However, the N-terminalsequence of peptides of 43 kDa and 41 kDa produced by trypsin treatmentof ZPI were the same, NLELGLTQSFAFIHKDFDV, SEQ ID NO: 5, and showed 75%identity (16 of 20 residues) with an amino acid sequence predicted bythe previously reported rat regeneration-associated serpin-1 (rasp-1)cDNA (19). Oligonucleotide primers based on rasp-1 cDNA sequence wereused as PCR primers and a human fetal liver cDNA library (Stratagene)was used as a template to produce a ˜330 bp probe for the subsequentisolation of ZPI cDNA (see Methods). Twenty-one positive plaquescontaining inserts of four different sizes were isolated from a humanliver cDNA library (Clontech). The nucleotide sequence, SEQ ID NO: 7,and predicted amino acid sequence, SEQ ID NO: 8 of the longest ZPI cDNAinsert, is shown in FIG. 6. Restriction mapping and limited sequenceanalysis of clones representative of the three shorter ZPI cDNA insertsizes suggest they are 5′ truncated forms of the cDNA shown.

The 5′ portion of the 2.44 kb ZPI cDNA contains six potential ATGtranslation start sites at nucleotides 156, 243, 259, 312, 347, and 467.The open reading frames following the first four ATG's encode 11, 22, 3,and 52 amino acids, respectively, before encountering stop codons. ATG₅(underlined with dashes in FIG. 6) is in the same reading frame as ATG₆and translation initiation at ATG₅ would add the forty amino acidsequence MSRSTQELLGYHCRLQDKLQEQEGSLAAEGRHSLASAADH, SEQ ID NO: 6, to theencoded protein.

Flanking nucleotides about the ATG codons, including ATG₅ and ATG₆,produce sequences that are not optimal for the initiation of translation(22). Nevertheless, ATG₆ is depicted as the initiator codon in FIG. 6because additional tests (see below) showed it was sufficient for ZPIexpression. On Northern analysis of a human multiple tissue blot, ˜2.4kb ZPI mRNA was strongly detected in liver, but undetectable in heart,lung, brain, spleen, testes and kidney (FIG. 7), suggesting that theliver is a major source of ZPI in vivo.

As depicted, the ZPI cDNA contains a 1335 bp open reading frame encodinga deduced protein of 444 amino acids. The predicted amino acid sequencehas a typical 21 residue signal peptide that is followed by theN-terminal sequence of the purified ZPI protein. Five potential N-linkedglycosylation sites are present. The nucleotide and predicted amino acidsequence of human ZPI are respectively 75% and 78% identical with thoseof rat rasp-1, suggesting that ZPI represents the human homologue ofthis rat protein.

The ZPI amino acid sequence is also 25-35% homologous with other membersof the serpin superfamily of protease inhibitors, includingα₁-antitrypsin, antithrombin, heparin cofactor II and protease nexin-1.The C-terminal region of ZPI shows the greatest similarity with theother members of the serpin superfamily, whereas the sequence of theN-terminal region of ZPI, which contains a very acidic domain (residues26-43, FIG. 6), does not show significant homology with these otherserpins.

The C-terminal amino acid sequences of ZPI, rasp-1, and certain otherserpins are shown in FIG. 8. Based on this alignment, the putative P1residue at the reactive centers of human ZPI and rat rasp-1 is atyrosine. Antitrypsin-related sequence (A1AU), an apparentlynon-transcribed DNA sequence highly homologous to that of antitrypsinand physically linked to the antitrypsin gene, also contains an aromaticresidue (tryptophan) at the P₁ site (23,24). In common with many otherserpins, the P₁′ residue in ZPI is a serine, whereas the P₁′ residue inrasp-1 is a cysteine.

Expression of Recombinant ZPI

To confirm that the protein encoded by the isolated cDNA possesses ZPIactivity and to determine the importance of Y387 to ZPI function,rZPI(WT) and two altered forms of ZPI, rZPI(Y387A) and rZPI(Y387R), wereexpressed in Chine hamster ovary (CHO) cells. Western blot analysis ofthe respective serum-free conditioned medias showed that wild type andthe altered forms of rZPI were present at similar concentrations (FIG.9). However, while rZPI(WT) and rZPI(Y387A) migrated with the sameapparent molecular mass as plasma-derived ZPI (72,000 Da), the bulk ofrZPI(Y387R) migrated with a molecular mass of 68,000 Da. Attempts toreduce the apparent proteolytic degradation of rZPI(Y387R) by includingaprotinin and soybean trypsin inhibitor in the serum-free culture mediaand adding a protease inhibitor cocktail to the collected conditionedmedia were unsuccessful (see Methods).

In a two-stage assay of PZ-dependent factor Xa inhibition, theserum-free conditioned media containing rZPI(WT) possessed substantialZPI activity (375 mU/mL), whereas conditioned media containingrZPI(Y387A) lacked activity (<10 mU/mL) and conditioned media containingrZPI(387R) had markedly reduced activity (21 mU/mL) (FIG. 9).

Based on oligonucleotide and amino acid sequence homology, ZPI appearsto be the human counterpart of rat rasp-1. Rasp-1 was initiallyidentified as a gene whose transcription is increased 3- to 4-foldfollowing subtotal hepatectomy in rats (19). However, rasp-1 expressionis increased to a similar extent in sham-operated rats, suggesting thatrasp-1 may be involved in the acute phase response. The rasp-1 geneproduct circulates in rat plasma with a reported molecular mass of˜50,000, whereas the molecular mass of plasma ZPI is ˜72,000 (17,19).This apparent difference in the molecular size between the rat and humangene-products could be related to the extent of glycosylation.Constitutive expression of both rasp-1 (19) and ZPI genes is high in theliver and not detectable in brain, heart, lung, kidney, spleen, andtestes by Northern analysis.

The ZPI cDNA is 2.44 kb in length and consistent with the smallesthybridizing species of ˜2.4 kb noted in liver on Northern analysis (FIG.7). Hybridizing bands of greater size likely represent incompletelyprocessed forms of ZPI MRNA. The 5′ region of the ZPI CDNA is relativelylong (466 bp) and contains several potential ATG translation startcodons. Four of these putative start codons are followed by terminationcodons, but the fifth ATG at nucleotide 347 is inframe with the ATG atnucleotide 467 that is tentatively designated as the authentic startcodon. All these potential ATG initiation start sites are flanked byless than ideal nucleotide sequences (22). The long 5′ untranslatedregion, the presence of multiple upstream AUG codons that encode smallopen reading frames, and the lack of an optimal initiation sequencecould all serve to suppress ZPI mRNA translation (22,25,26). Whetherthis is true, and whether an alternative form of ZPI is produced throughtranslation initiation at the fifth AUG (nt 347), will require directtesting.

ZPI has 25-35% overall homology with other members of the serpinsuperfamily and its primary structure contains 40 of the 5′ residuespreviously designated as essential for serpin tertiary structure (27).These conserved residues reside in the apolar core and the spine ofserpin molecules. Amino acid alignment of ZPI and rasp-1 with otherserpins suggests that the P₁ residue at their reactive centers is atyrosine, which would set them apart from other serpins.

To confirm the role of Y387 in the inhibition of factor Xa by ZPI,altered forms of ZPI in which this residue was changed to an alanine orarginine were evaluated. rZPI(Y387A) was stable under the tissue cultureconditions required for its expression and lacked PZ-dependentanti-factor Xa activity. In contrast to rZPI(WT), rZPI(Y387R) wasapparently proteolytically degraded during the production of conditionedmedia despite the use of multiple protease inhibitors. The proteolyticevent reduces the mass of ZPI by ˜4,000 Da consistent with cleavageoccurring following R387, but the enzyme(s) responsible for thisproteolysis is not known.

In sum, the tests with rZPI(Y387A) and rZPI(Y387R) suggest that Y387 iscritical for PZ-dependent factor Xa inhibition and are consistent withthe notion that Y387 is the P₁ residue at the reactive center of ZPI.

The coagulation inhibitors PZ, ZPI and combination of PZ and ZPI can beadministered to a patient by conventional means, preferably informulations with pharmaceutically acceptable diluents and carriers. Theamount of the active component in the formulation which is anadministered must be an effective amount, i.e., an amount which issufficient to inhibit coagulation. Parenteral administration of theactive component such as in physiologic saline, buffered saline, e.g.phosphate-buffered saline (PBS), HEPES buffer and the like buffers, areillustrative. Other suitable formulations of the active component inpharmaceutically acceptable diluents and carriers in therapeutic dosageform can be prepared by reference to numerous general texts in the fieldwell-known to the person skilled in the art, e.g., Remington'sPharmaceutical Sciences, Ed. Arthur Osol, 16th ed. 1980, Mack PublishingCo., Easton, Pa., and 18th ed. 1990.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

TABLE I Apparent inhibition of factor Xa produced by incubation with PZ,cephalin and calcium ions before one- stage assay. PZ_(r) refers tothrombin-treated PZ (see Methods). Incubation Period (sec.) ApparentFactor Xa PZ Inhibition (%) 120 0 0 120 15 50 120 30 61 120 60 72 120 9076 120 120 78 120 PZ_(r) 120 0 15 120 70 30 120 71 60 120 73 90 120 76120 120 78 120 120 w/o cephalin 0 120 120 w/o Ca++ 0

TABLE II ZPI PURIFICATION Specific Purifica- Volume Protein ActivityAct. tion- Yield Step mL mg* units ⁺ units/mg fold % Plasma 2300 144,740—# — — — Barium 1430 59,730 1820 0.031 1.0 100 Absorption, NH₄SO₄fractionation PEG 257 25,950 1720 0.066 2.2 94 fractionation S-FastFlow. 25 191 1365 7.15 231 75 Concen- tration Mono-Q 10 50.2 1056 21.0677 58 Heparin- 5.8 1.32 837 634 20.450 46 Sepharose Mono-S 1.9 0.59 5901000 32,800 32 *Protein determined assuming A₂₈₀ 1.0 = 1.0 mg/mL⁺Activity expressed in abitrary units with 1.0 unit = 1 ug purified ZPI#Activity of plasma could not be determined due to thrombin generationin first stage of functional assay

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SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 16 <210> SEQ ID NO 1 <211>LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <221> NAME/KEY: Xaa=Unknown amino acid <222> LOCATION:Xaa=Indeterminate residues 10 & 11; can be any amino acid <223> OTHERINFORMATION: /note=“synthetic construct” <400> SEQUENCE: 1 Leu Ala ProSer Pro Gln Ser Pro Glu Xaa Xaa Ala 1 5 10 <210> SEQ ID NO 2 <211>LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <221> NAME/KEY: Xaa=Unknown amino acid <222> LOCATION:Xaa=Indeterminate residue 13; can be any amino acid <223> OTHERINFORMATION: /note=“synthetic construct” <400> SEQUENCE: 2 Arg Tyr LysGly Gly Ser Pro Xaa Ile Ser Gln Pro Xaa Leu 1 5 10 <210> SEQ ID NO 3<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: /note=“synthetic construct”<400> SEQUENCE: 3 acccagggta gctttgcctt cat 23 <210> SEQ ID NO 4 <211>LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: /note=“synthetic construct” <400>SEQUENCE: 4 gtacatcatg ggcaccttaa c 21 <210> SEQ ID NO 5 <211> LENGTH:19 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: /note=“synthetic construct” <400> SEQUENCE: 5Asn Leu Glu Leu Gly Leu Thr Gln Ser Phe Ala Phe Ile His Lys 1 5 10 15Asp Phe Asp Val <210> SEQ ID NO 6 <211> LENGTH: 40 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:/note=“synthetic construct” <400> SEQUENCE: 6 Met Ser Arg Ser Thr GlnGlu Leu Leu Gly Tyr His Cys Arg Leu 1 5 10 15 Gln Asp Lys Leu Gln GluGln Glu Gly Ser Leu Ala Ala Glu Gly 20 25 30 Arg His Ser Leu Ala Ser AlaAla Asp His 35 40 <210> SEQ ID NO 7 <211> LENGTH: 2466 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: /note=“synthetic construct” <400> SEQUENCE: 7 ctggagtggggtaagaggcg aattatagac acaaggggct cctctgcagg 50 aaggaggcca agggaaagaggcttgaaagg cttgatattt cacccaccac 100 cactcactgc cggagtaagc aggtctccccttcccagggc tgaggggagg 150 cagggatgtg tgctgtccca gggctgagaa gtggcaggtgagctggtgat 200 tccttactgc ccaggttcgt tctaggaagg tgcgtcctca ccatgctgga250 tggtgtccta gtccaggagc accccctgag ctcctggcct agactccaaa 300gggttgggta gatgagcaaa gactttacaa agaccttagg cgatatatgt 350 ccaggagcacccaggaatta ctgggctacc actgcagact gcaggacaag 400 ctccaagaac aggaaggaagtcttgcagct gaagggaggc actccttggc 450 ctccgcagcc gat cac atg aag gtg gtgcca agt ctc ctg ctc 493 Met Lys Val Val Pro Ser Leu Leu Leu -20 -15 tccgtc ctc ctg gca cag gtg tgg ctg gta ccc ggc ttg gcc 535 Ser Val Leu LeuAla Gln Val Trp Leu Val Pro Gly Leu Ala -10 -5 -1 1 ccc agt cct cag tcgcca gag acc cca gcc cct cag aac cag 577 Pro Ser Pro Gln Ser Pro Glu ThrPro Ala Pro Gln Asn Gln 5 10 15 acc agc agg gta gtg cag gct ccc aag gaggaa gag gaa gat 619 Thr Ser Arg Val Val Gln Ala Pro Lys Glu Glu Glu GluAsp 20 25 30 gag cag gag gcc agc gag gag aag gcc agt gag gaa gag aaa 661Glu Gln Glu Ala Ser Glu Glu Lys Ala Ser Glu Glu Glu Lys 35 40 gcc tggctg atg gcc agc agg cag cag ctt gcc aag gag act 703 Ala Trp Leu Met AlaSer Arg Gln Gln Leu Ala Lys Glu Thr 45 50 55 tca aac ttc gga ttc agc ctgctg cga aag atc tcc atg agg 745 Ser Asn Phe Gly Phe Ser Leu Leu Arg LysIle Ser Met Arg 60 65 70 cac gat ggc aac atg gtc ttc tct cca ttt ggc atgtcc ttg 787 His Asp Gly Asn Met Val Phe Ser Pro Phe Gly Met Ser Leu 7580 85 gcc atg aca ggc ttg atg ctg ggg gcc aca ggg ccg act gaa 829 AlaMet Thr Gly Leu Met Leu Gly Ala Thr Gly Pro Thr Glu 90 95 100 acc cagatc aag aga ggg ctc cac ttg cag gcc ctg aag ccc 871 Thr Gln Ile Lys ArgGly Leu His Leu Gln Ala Leu Lys Pro 105 110 acc aag ccc ggg ctc ctg ccttcc ctc ttt aag gga ctc aga 913 Thr Lys Pro Gly Leu Leu Pro Ser Leu PheLys Gly Leu Arg 115 120 125 gag acc ctc tcc cgc aac ctg gaa ctg ggc ctcaca cag ggg 955 Glu Thr Leu Ser Arg Asn Leu Glu Leu Gly Leu Thr Gln Gly130 135 140 agt ttt gcc ttc atc cac aag gat ttt gat gtc aaa gag act 997Ser Phe Ala Phe Ile His Lys Asp Phe Asp Val Lys Glu Thr 145 150 155 ttcttc aat tta tcc aag agg tat ttt gat aca gag tgc gtg 1039 Phe Phe Asn LeuSer Lys Arg Tyr Phe Asp Thr Gly Cys Val 160 165 170 cct atg aat ttt cgcaat gcc tca cag gcc aaa agg ctc atg 1081 Pro Met Asn Phe Arg Asn Ala SerGln Ala Lys Arg Leu Met 175 180 aat cat tac att aac aaa gag act cgg gggaaa att ccc aaa 1123 Asn His Tyr Ile Asn Lys Glu Thr Arg Gly Lys Ile ProLys 185 190 195 ctg ttt gat gag att aat cct gaa acc aaa tta att ctt gtg1165 Leu Phe Asp Glu Ile Asn Phe Glu Thr Lys Leu Ile Leu Val 200 205 210gat tac atc ttg ttc aaa ggg aaa tgg ttg acc cca ttt gac 1207 Asp Tyr IleLeu Phe Lys Gly Lys Trp Leu Thr Pro Phe Asp 215 220 225 cct gtc ttc accgaa gtc gac act ttc cac ctg gac aag tac 1249 Pro Val Phe Thr Glu Val AspThr Phe His Leu Asp Lys Tyr 230 235 240 aag acc att aag gtg ccc atg atgtac ggt gca ggc aag ttt 1291 Lys Thr Ile Lys Val Pro Met Met Tyr Gly AlaGly Lys Phe 245 250 gcc tcc acc ttt gac aag aat ttt cgt tgt cat gtc ctcaaa 1333 Ala Ser Thr Phe Asp Lys Asn Phe Arg Cys His Val Leu Lys 255 260265 ctg ccc tac caa gga aat gcc acc atg ctg gtg gtc ctc atg 1375 Leu ProTyr Gln Gly Asn Ala Thr Met Leu Val Val Leu Met 270 275 280 gag aaa atgggt gac cac ctc gcc ctt gaa gac tac ctg acc 1417 Glu Lys Met Gly Asp HisLeu Ala Leu Glu Asp Tyr Leu Thr 285 290 295 aca gac ttg gtg gag aca tggctc aga aac atg aaa acc aga 1459 Thr Asp Leu Val Glu Thr Trp Leu Arg AsnMet Lys Thr Arg 300 305 310 aac atg gaa gtt ttc ttt ccg aag ttc aag ctagat cag aag 1501 Asn Met Glu Val Phe Phe Pro Lys Phe Lys Leu Asp Gln Lys315 320 tat gag atg cat gag ctg ctt agg cag atg gga atc aga aga 1543 TyrGlu Met His Glu Leu Leu Arg Gln Met Gly Ile Arg Arg 325 330 335 atc ttctca ccc ttt gct gac ctt agt gaa ctc tca gct act 1585 Ile Phe Ser Pro PheAla Asp Leu Ser Glu Leu Ser Ala Thr 340 345 350 gga aga aat ctc caa gtatcc agg gtt tta caa aga aca gtg 1627 Gly Arg Asn Leu Gln Val Ser Arg ValLeu Gln Arg Thr Val 355 360 365 att gaa gtt gat gaa agg ggc act gag gcagtg gca gga atc 1669 Ile Glu Val Asp Glu Arg Gly Thr Glu Ala Val Ala GlyIle 370 375 380 ttg tca gaa att act gct tat tcc atg cct cct gtc atc aaa1711 Leu Ser Glu Ile Thr Ala Tyr Ser Met Pro Pro Val Ile Lys 385 390 gtggac cgg cca ttt cat ttc atg atc tat gaa gaa acc tct 1753 Val Asp Arg ProPhe His Phe Met Ile Tyr Glu Glu Thr Ser 395 400 405 gga atg ctt ctg tttctg ggc agg gtg gtg aat ccg act ctc 1795 Gly Met Leu Leu Phe Leu Gly ArgVal Val Asn Pro Thr Leu 410 415 420 cta taa ttcaggacac gcataagcacttcgcgtgta gtagatgctg 1841 Leu 423 aatctgaggt atcaaacaca cacaggataccagcaatgga tggcagggga 1891 gagtgttcct tttgttctta actagtttag ggtgttctcaaataaataca 1941 gtagtcccca cttatctgag ggggatacat tcaaagaccc ccagcagatg1991 cctgaaacgg tggacagtgc tgaaccttat atatattttt tcctacacat 2041acatacctat gataaagttt aatttataaa ttaggcacag taagagatta 2091 acaataataacaacattaag taaaatgagt tacttgaatg caagcactgc 2141 aataccataa cagtcaaactgattatagag aaggctacta agtgactcat 2191 gggcgaggag catagacagt gtggagacattgggcaaggg gagaattcac 2241 atcctgggtg ggacagagca ggacaatgca agattccatcccactactca 2291 gaatggcatg ctgcttaaga cttttagatt gtttatttct ggaatttttc2341 atttaatgtt tttggaccat ggttgaccat ggttaactga gactgcagaa 2391agcaaaacca tggataaggg aggactacta caaaagcatt aaattgatac 2441 atattttttaaaaaaaaaaa aaaaa 2466 <210> SEQ ID NO 8 <211> LENGTH: 444 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: /note=“synthetic construct” <400> SEQUENCE: 8 Met Lys ValVal Pro Ser Leu Leu Leu Ser Val Leu Leu Ala -20 -15 -10 Val Val Trp LeuVal Pro Gly Leu Ala Pro Ser Pro Gln Ser -5 -1 1 5 Pro Glu Thr Pro AlaPro Gln Asn Gln Thr Ser Arg Val Val 10 15 20 Gln Ala Pro Lys Glu Glu GluGlu Asp Glu Gln Glu Ala Ser 25 30 35 Glu Glu Lys Ala Ser Glu Glu Glu LysAla Trp Leu Met Ala 40 45 Ser Arg Gln Gln Leu Ala Lys Glu Thr Ser AsnPhe Gly Phe 50 55 60 Ser Leu Leu Arg Lys Ile Ser Met Arg His Asp Gly AsnMet 65 70 75 Val Phe Ser Pro Phe Gly Met Ser Leu Ala Met Thr Gly Leu 8085 90 Met Leu Gly Ala Thr Gly Pro Thr Glu Thr Gln Ile Lys Arg 95 100 105Gly Leu His Leu Gln Ala Leu Lys Pro Thr Lys Pro Gly Leu 110 115 Leu ProSer Leu Phe Lys Gly Leu Arg Glu Thr Leu Ser Arg 120 125 130 Asn Leu GluLeu Gly Leu Thr Gln Gly Ser Phe Ala Phe Ile 135 140 145 His Lys Asp PheAsp Val Lys Glu Thr Phe Phe Asn Leu Ser 150 155 160 Lys Arg Tyr Phe AspThr Gly Cys Val Pro Met Asn Phe Arg 165 170 175 Asn Ala Ser Gln Ala LysArg Leu Met Asn His Tyr Ile Asn 180 185 Lys Glu Thr Arg Gly Lys Ile ProLys Leu Phe Asp Glu Ile 190 195 200 Asn Phe Glu Thr Lys Leu Ile Leu ValAsp Tyr Ile Leu Phe 205 210 215 Lys Gly Lys Trp Leu Thr Pro Phe Asp ProVal Phe Thr Glu 220 225 230 Val Asp Thr Phe His Leu Asp Lys Tyr Lys ThrIle Lys Val 235 240 245 Pro Met Met Tyr Gly Ala Gly Lys Phe Ala Ser ThrPhe Asp 250 255 Lys Asn Phe Arg Cys His Val Leu Lys Leu Pro Tyr Gln Gly260 265 270 Asn Ala Thr Met Leu Val Val Leu Met Glu Lys Met Gly Asp 275280 285 His Leu Ala Leu Glu Asp Tyr Leu Thr Thr Asp Leu Val Glu 290 295300 Thr Trp Leu Arg Asn Met Lys Thr Arg Asn Met Glu Val Phe 305 310 315Phe Pro Lys Phe Lys Leu Asp Gln Lys Tyr Glu Met His Glu 320 325 Leu LeuArg Gln Met Gly Ile Arg Arg Ile Phe Ser Pro Phe 330 335 340 Ala Asp LeuSer Glu Leu Ser Ala Thr Gly Arg Asn Leu Gln 345 350 355 Val Ser Arg ValLeu Gln Arg Thr Val Ile Glu Val Asp Glu 360 365 370 Arg Gly Thr Glu AlaVal Ala Gly Ile Leu Ser Glu Ile Thr 375 380 385 Ala Tyr Ser Met Pro ProVal Ile Lys Val Asp Arg Pro Phe 390 395 His Phe Met Ile Tyr Glu Glu ThrSer Gly Met Leu Leu Phe 400 405 410 Leu Gly Arg Val Val Asn Pro Thr LeuLeu 415 420 <210> SEQ ID NO 9 <211> LENGTH: 53 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:/note=“synthetic construct” <400> SEQUENCE: 9 Glu Arg Gly Thr Glu AlaVal Ala Gly Ile Leu Ser Glu Ile Thr 1 5 10 15 Ala Tyr Ser Met Pro ProVal Ile Lys Val Asp Arg Pro Phe His 20 25 30 Phe Met Ile Tyr Glu Glu ThrSer Gly Met Leu Leu Phe Leu Gly 35 40 45 Arg Val Val Asn Pro Thr Leu Leu50 <210> SEQ ID NO 10 <211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:/note=“synthetic construct” <400> SEQUENCE: 10 Glu Arg Gly Thr Glu ValVal Ser Gly Thr Val Ser Glu Ile Thr 1 5 10 15 Ala Tyr Cys Met Pro ProVal Ile Lys Val Asp Arg Pro Phe His 20 25 30 Phe Ile Ile Tyr Glu Glu MetSer Arg Met Leu Leu Phe Leu Gly 35 40 45 Arg Val Val Asn Pro Thr Val Leu50 <210> SEQ ID NO 11 <211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:/note=“synthetic construct” <400> SEQUENCE: 11 Glu Lys Gly Thr Glu AlaAla Gly Ala Met Phe Leu Glu Ala Ile 1 5 10 15 Pro Met Ser Ile Pro ProGlu Val Lys Phe Asn Lys Pro Phe Val 20 25 30 Phe Leu Met Ile Glu Gln AsnThr Lys Ser Pro Leu Phe Met Gly 35 40 45 Lys Val Val Asn Pro Thr Gln Lys50 <210> SEQ ID NO 12 <211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:/note=“synthetic construct” <400> SEQUENCE: 12 Glu Lys Gly Thr Glu AlaThr Gly Ala Pro His Leu Glu Glu Lys 1 5 10 15 Ala Trp Ser Lys Tyr GlnThr Val Met Phe Asn Arg Pro Phe Leu 20 25 30 Val Ile Ile Lys Glu Tyr IleThr Asn Phe Pro Leu Phe Ile Gly 35 40 45 Lys Val Val Asn Pro Thr Gln Lys50 <210> SEQ ID NO 13 <211> LENGTH: 56 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:/note=“synthetic construct” <400> SEQUENCE: 13 Glu Glu Gly Ser Glu AlaAla Ala Ser Thr Ala Val Val Ile Ala 1 5 10 15 Gly Arg Ser Leu Asn ProAsn Arg Val Thr Phe Lys Ala Asn Arg 20 25 30 Pro Phe Leu Val Phe Ile ArgGlu Val Pro Leu Asn Thr Ile Ile 35 40 45 Phe Met Gly Arg Val Ala Asn ProCys Val Lys 50 55 <210> SEQ ID NO 14 <211> LENGTH: 53 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: /note=“synthetic construct” <400> SEQUENCE: 14 Glu Glu GlyThr Gln Ala Thr Thr Val Thr Thr Val Gly Phe Met 1 5 10 15 Pro Leu SerThr Gln Val Arg Phe Thr Val Asp Arg Pro Phe Leu 20 25 30 Phe Leu Ile TyrGlu His Arg Thr Ser Cys Leu Leu Phe Met Gly 35 40 45 Arg Val Ala Asn ProSer Arg Ser 50 <210> SEQ ID NO 15 <211> LENGTH: 50 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:/note=“synthetic construct” <400> SEQUENCE: 15 Glu Asp Gly Thr Lys AlaSer Ala Ala Thr Thr Ala Ile Leu Ile 1 5 10 15 Ala Arg Ser Ser Pro ProTrp Phe Ile Val Asp Arg Pro Phe Leu 20 25 30 Phe Phe Ile Arg His Asn ProThr Gly Ala Val Leu Phe Met Gly 35 40 45 Gln Ile Asn Lys Pro 50 <210>SEQ ID NO 16 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: /note=“syntheticconstruct” <400> SEQUENCE: 16 Leu Ala Pro Ser Pro Gln Ser Pro Glu ThrPro Ala 1 5 10

What is claimed is:
 1. A method of inhibiting factor Xa formation in apatient in need thereof comprising administering to said patient acombination of human protein Z and ZPI in an amount sufficient toinhibit factor Xa formation.
 2. The method of claim 1 in which the ZPIhas residues 1-423 of SEQ ID NO:8.